Disruption of embryonic ROCK signaling reproduces the sarcomeric phenotype of hypertrophic cardiomyopathy
Kate E. Bailey, Guy A. MacGowan, Simon Tual-Chalot, Lauren Phillips, Timothy J. Mohun, Deborah J. Henderson, Helen M. Arthur, Simon D. Bamforth, Helen M. Phillips
Kate E. Bailey, Guy A. MacGowan, Simon Tual-Chalot, Lauren Phillips, Timothy J. Mohun, Deborah J. Henderson, Helen M. Arthur, Simon D. Bamforth, Helen M. Phillips
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Research Article Cardiology

Disruption of embryonic ROCK signaling reproduces the sarcomeric phenotype of hypertrophic cardiomyopathy

Abstract

Sarcomeric disarray is a hallmark of gene mutations in patients with hypertrophic cardiomyopathy (HCM). However, it is unknown when detrimental sarcomeric changes first occur and whether they originate in the developing embryonic heart. Furthermore, Rho kinase (ROCK) is a serine/threonine protein kinase that is critical for regulating the function of several sarcomeric proteins, and therefore, our aim was to determine whether disruption of ROCK signaling during the earliest stages of heart development would disrupt the integrity of sarcomeres, altering heart development and function. Using a mouse model in which the function of ROCK is specifically disrupted in embryonic cardiomyocytes, we demonstrate a progressive cardiomyopathy that first appeared as sarcomeric disarray during cardiogenesis. This led to abnormalities in the structure of the embryonic ventricular wall and compensatory cardiomyocyte hypertrophy during fetal development. This sarcomeric disruption and hypertrophy persisted throughout adult life, triggering left ventricular concentric hypertrophy with systolic dysfunction, and reactivation of fetal gene expression and cardiac fibrosis, all typical features of HCM. Taken together, our findings establish a mechanism for the developmental origin of the sarcomeric phenotype of HCM and suggest that variants in the ROCK genes or disruption of ROCK signaling could, in part, contribute to its pathogenesis.

Authors

Kate E. Bailey, Guy A. MacGowan, Simon Tual-Chalot, Lauren Phillips, Timothy J. Mohun, Deborah J. Henderson, Helen M. Arthur, Simon D. Bamforth, Helen M. Phillips

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Figure 5

Destabilization of sarcomeres in ROCKDNGata5-Cre mutant cardiomyocytes at E10.5. (A–J) Transverse sections from E10.5 embryos were stained by immunofluorescence to identify all cardiomyocytes (cTnI staining, green) and Cre-activated cardiomyocytes/GFP+ cells (red staining).

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Destabilization of sarcomeres in ROCKDNGata5-Cre mutant cardiomyocytes a...
In Cre-activated cardiomyocytes of control embryos, cTnI was cortical (red arrows in B), confirmed by the 2 separate curves in the fluorescence intensity profile plot (E). In contrast, in Cre-activated cardiomyocytes of ROCKDNGata5-Cre mutants, cTnI staining was disrupted, as it was diffuse throughout the cell (red arrows in D), confirmed by the continuous line in the fluorescence intensity profile plot (F). The cTnI staining in GFP negative cardiomyocytes in control and ROCKDNGata5-Cre mutants was identical (white arrows in G–J). n=6 for each genotype. (K–R) Representative TEM imaging of myofibrils at E10.5. In controls, the sarcomeres generally align in close proximity to the plasma membrane and are highly organized (arrows in K). In ROCKDNGata5-Cre cardiomyocytes, the myofibrils are located throughout the cells and show sarcomere collapse (arrows in L). The average width of the Z discs throughout the sarcomeres was significantly increased in ROCKDNGata5-Cre cardiomyocytes compared with controls (M, N, and U). Myofibril discontinuity was present at cellular junctions in ROCKDNGata5-Cre as the myofibrils approached the intercalated discs at more varied wider angles (arrows in P and V) but not seen in controls (O). Areas of G-actin accumulation were detected in ROCKDNGata5-Cre cardiomyocytes (R) more frequently that in control hearts (Q) giving a significant increase in G-actin density (X). (S) There was a significant increase in sarcomere length and decrease in sarcomere width in ROCKDNGata5-Cre mutants compared with sarcomeres in control hearts. (T) The number of repeating sarcomeric units, per field of view in comparative regions, was significantly reduced in ROCKDNGata5-Cre cardiomyocytes. (W) As a consequence of the abnormal sarcomeric structure, in ROCKDNGata5-Cre cardiomyocytes, there was significantly reduced myofibril density per field of view. (X) The percent area containing G-actin, per field of view, showed a significant increase in ROCKDNGata5-Cre cardiomyocytes. n = 5 for each genotype. lv, left ventricle; pm, plasma membrane. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired t test. Scale bars: 50 μm (A–D and G–J) and 500 nm (K–R).